The invention relates to genes involved in the onset of muscular dystrophy.
Muscular dystrophies constitute a heterogeneous group of disorders. Most are characterized by weakness and atrophy of the proximal muscles, although in rare myopathies such as xe2x80x9cMiyoshi myopathyxe2x80x9d symptoms may first arise in distal muscles. Of the various hereditary types of muscular dystrophy, several are caused by mutations or deletions in genes encoding individual components of the dystrophin-associated protein (DAP) complex. It is this DAP complex that links the cytoskeletal protein dystrophin to the extracellular matrix protein, laminin-2.
Muscular dystrophies may be classified according to the gene mutations that are associated with specific clinical syndromes. For example, mutations in the gene encoding the cytoskeletal protein dystrophin result in either Duchenne""s Muscular Dystrophy or Becker""s Muscular Dystrophy, whereas mutations in the gene encoding the extracellular matrix protein merosin produce Congenital Muscular Dystrophy. Muscular dystrophies with an autosomal recessive mode of inheritance include xe2x80x9cMiyoshi myopathyxe2x80x9d and the several limb-girdle muscular dystrophies (LGMD2). Of the limb-girdle muscular dystrophies, the deficiencies resulting in LGMD2C, D, E, and F result from mutations in genes encoding the membrane-associated sarcoglycan components of the DAP complex.
A novel protein, designated dysferlin, is identified and characterized. The dysferlin gene is normally expressed in skeletal muscle cells and is selectively mutated in several families with the hereditary muscular dystrophies, e.g., Miyoshi myopathy (MM) and limb girdle muscular dystrophy-2B (LGMD2B). These characteristics of dysferlin render it a candidate disease gene for both MM and LGMD2B. An additional novel protein, brain-specific dysferlin, has also been identified. Defects in brain-specific dysferlin may predispose to selected disorders of the central nervous system. Moreover, the expression of brain-specific dysferlin may be important as a marker for normal neural development (e.g., in vivo or in neural cells in culture). Manipulation of levels of expression of brain-specific dysferlin, and of the type of expressed brain-specific dysferlin is of use for analyzing the function of brain-specific dysferlin and related dysferlin-associated molecules.
The invention features an isolated DNA which includes a nucleotide sequence hybridizing under stringent hybridization conditions to a strand of SEQ ID NO:3 or SEQ ID NO:117. SEQ ID NO:117 corresponds to nucleotides 374-6613 of wild type dysferlin.
The invention also features an isolated DNA including a nucleotide sequence selected from SEQ ID NOs:4-12. SEQ ID NOs:4-12 are oligonucleotides that span the mutations of 537insA, Q605X, 5966delG, E1883X, 6391+1G to A, I1298V, R2042C, H1857R, and 6071/2delAG, respectively (Table 2).
Also within the invention is an isolated DNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs:22-30.
Also within the invention is an isolated DNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs:22-30. SEQ ID NOs:22-30 are oligonucleotides with wild type sequences that span the mutant regions identified in the mutants 537inSA, Q605X, 5966delG, E1883X, 6391+1G to A, I1298V, R2042C, H1857R, and 6071/2delAG, respectively (Table 2).
Also within the invention is a pair of PCR primers consisting of:
(a) a first single stranded oligonucleotide consisting of 14-50 contiguous nucleotides of the sense strand of SEQ ID NO:117; and
(b) a second single stranded oligonucleotide consisting of 14-50 contiguous nucleotides of the antisense strand of SEQ ID NO:117, wherein the sequence of at least one of the oligonucleotides is identical to a portion of a strand of SEQ ID NO:3, and the first oligonucleotide is not complementary to the second oligonucleotide.
Also within the invention is a pair of single stranded oligonucleotides selected from of SEQ ID NOs 130-231, SEQ ID NO:110, and SEQ ID NO:112.
Also within the invention is an isolated DNA including a nucleotide sequence that encodes a protein that shares at least 70% sequence identity with SEQ ID NO:2, or a complement of the nucleotide sequence.
Also within the invention is an isolated DNA including a nucleotide sequence which hybridizes under stringent hybridization conditions to a strand of a nucleic acid, the nucleic acid having a sequence selected from SEQ ID NOs:31-79 and 90-100. SEQ ID NOs:90-100 are intron sequences from a dysferlin gene. Specifically, SEQ ID NOs:90-100 are intron sequence 5xe2x80x2 of exon 50, intron sequence 3xe2x80x2 of exon 50, intron sequence 5xe2x80x2 of exon 51, intron sequence 3xe2x80x2 of exon 51, intron sequence 5xe2x80x2 of exon 52, intron sequence 3xe2x80x2 of exon 52, intron sequence 5xe2x80x2 of exon 53, intron sequence 3xe2x80x2 of exon 53, intron sequence 5xe2x80x2 of exon 54, intron sequence 3xe2x80x2 of exon 54, and intron sequence 5xe2x80x2 of exon 55.
Also within the invention is a single stranded oligonucleotide of 14-50 nucleotides in length having a nucleotide sequence which is identical to a portion of a strand of a nucleic acid selected from SEQ ID NOs:31-79 and 90-100.
Also within the invention is a pair of PCR primers consisting of:
(a) a first single stranded oligonucleotide consisting of 14-50 contiguous nucleotides of the sense strand of a nucleic acid selected from SEQ ID NOs:31-85; and
(b) a second single stranded oligonucleotide consisting of 14-50 contiguous nucleotides of the antisense strand of a nucleic acid selected from SEQ ID NOs:31-85, wherein the sequence of at least one of the oligonucleotides includes a sequence identical to a portion of a strand of a nucleic acid selected from SEQ ID NOs: 31-79 and 90-100, and the first oligonucleotide is not complementary to the second oligonucleotide.
Also within the invention is a pair of single stranded oligonucleotides selected from SEQ ID NOs 101-116, SEQ ID NOs 184-185, SEQ ID NOs 188-191, SEQ ID NOs 210-213, and SEQ ID NOs 216-217.
Also within the invention is a substantially pure protein that has an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO:2.
Also within the invention is a substantially pure protein the sequence of which includes amino acid residues 1-500, 501-1000, 1001-1500, or 1501-2080 of SEQ ID NO:2.
Also within the invention is a substantially pure protein including the amino acid sequence of SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, or SEQ ID NO:89.
In another aspect, the invention features a transgenic non-human mammal having a transgene disrupting or interfering with the expression of a dysferlin gene, the transgene being chromosomally integrated into the germ cells of the animal.
Another embodiment of the invention features a method of decreasing the symptoms of muscular dystrophy in a mammal by introducing into a cell of the mammal (e.g., a muscle cell or a muscle precursor cell) an isolated DNA which hybridizes under stringent hybridization conditions to a strand of SEQ ID NO:3.
Another aspect of the invention provides a method for identifying a patient, a fetus, or a pre-embryo at risk for having a dysferlin-related disorder by (a) providing a sample of genomic DNA from the patient, fetus, or pre-embryo; and (b) determining whether the sample contains a mutation in a dysferlin gene.
In another aspect, the invention provides a method for identifying a patient, a fetus, or a pre-embryo at risk for having a dysferlin-related disorder by (a) providing a sample including dysferlin mRNA from the patient, fetus, or pre-embryo; and (b) determining whether the dysferlin mRNA contains a mutation.
Methods of identifying mutations in a dysferlin sequence are useful for predicting (e.g., predicting whether an individual is at risk for developing a dysferlin-related disorder) or diagnosing disorders associated with dysferlin, e.g., MM and LGMD2B. Such methods can also be used to determine if an individual, fetus, or a pre-embryo is a carrier of a dysferlin mutation, for example in screening procedures. Methods which distinguish between different dysferlin alleles (e.g., a mutant dysferlin allele and a normal dysferlin allele) can be used to determine carrier status.
The invention also features an isolated nucleic acid comprising a nucleotide sequence which hybridizes under stringent hybridization conditions to nucleic acids 3284-3720 of SEQ ID NO:232, or the complement of the nucleotide sequence. An isolated nucleic acid including a nucleotide sequence identical to the sequence of nucleotides 3284-3720 of SEQ ID NO:232, or a complement of the nucleotide sequence is also a feature of the invention. The isolated nucleic acid can include the entire sequence of SEQ ID NO:232 or the complement of SEQ ID NO:232.
Another aspect of the invention features an isolated polypeptide that includes: a) at least 15 contiguous amino acids of the polypeptide comprising amino acids 1-24 of SEQ ID NO:233, b) a naturally occuring allelic variant of a polypeptide comprising amino acids 1-24 of SEQ ID NO:233, or c) an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes under stringent conditions to nucleotides 3284-3720 of SEQ ID NO:232. The polypeptide of this aspect can include the entire sequence of SEQ ID NO:233.
Also included in the invention is a vector comprising the nucleic acid of claim 44 and a cell that contains the vector. Another aspect of the invention features a method of making a polypeptide by culturing the cell which contains the vector.
The invention also features an antibody which specifically binds to a polypeptide of such as those described above. The antibody can bind to a polypeptide selected from amino acids 253-403 of SEQ ID NO:233, amino acids 624-865 of SEQ ID NO:233, and amino acids 1664-1786 of SEQ ID NO:233. Antibodies of the invention can be monclonal or polyclonal antibodies.
An xe2x80x9cisolated DNAxe2x80x9d is DNA which has a naturally occurring sequence corresponding to part or all of a given gene but is free of the two genes that normally flank the given gene in the genome of the organism in which the given gene naturally occurs. The term therefore includes a recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote. It also includes a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment, as well as a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. The term excludes intact chromosomes and large genomic segments containing multiple genes contained in vectors or constructs such as cosmids, yeast artificial chromosomes (YACs), and P1-derived artificial chromosome (PAC) contigs.
A xe2x80x9cnoncoding sequencexe2x80x9d is a sequence which corresponds to part or all of an intron of a gene, or to a sequence which is 5xe2x80x2 or 3xe2x80x2 to a coding sequence and so is not normally translated.
An expression control sequence is xe2x80x9coperably linkedxe2x80x9d to a coding sequence when it is within the same nucleic acid and can control expression of the coding sequence.
A xe2x80x9cproteinxe2x80x9d or xe2x80x9cpolypeptidexe2x80x9d is any chain of amino acids linked by peptide bonds, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.
As used herein, the term xe2x80x9cpercent sequence identityxe2x80x9d means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. For purposes of the present invention, percent sequence identity between two polypeptides is to be determined using the Gap program and the default parameters as specified therein. The Gap program is part of the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705.
The algorithm of Myers and Miller, CABIOS (1989) can also be used to determine whether two sequences are similar or identical. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
As used herein, the term xe2x80x9cstringent hybridization conditionsxe2x80x9d means the following DNA hybridization and wash conditions: hybridization at 60xc2x0 C. in the presence of 6xc3x97SSC, 0.5% SDS, 5xc3x97Denhardt""s Reagent, and 100 xcexcg/ml denatured salmon sperm DNA; followed by a first wash at room temperature for 20 minutes in 0.5xc3x97SSC and 0.1% SDS and a second wash at 55xc2x0 C. for 30 minutes in 0.2xc3x97SSC and 0.1% SDS.
A xe2x80x9csubstantially pure proteinxe2x80x9d is a protein separated from components that naturally accompany it. The protein is considered to be substantially pure when it is at least 60%, by dry weight, free from the proteins and other naturally-occurring organic molecules with which it is naturally associated. Preferably, the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure dysferlin protein can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding a dysferlin polypeptide, or by chemical synthesis. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A chemically synthesized protein or a recombinant protein produced in a cell type other than the cell type in which it naturally occurs is, by definition, substantially free from components that naturally accompany it. Accordingly, substantially pure proteins include those having sequences derived from eukaryotic organisms but which have been recombinantly produced in E. coli or other prokaryotes.
An antibody that xe2x80x9cspecifically bindsxe2x80x9d to an antigen is an antibody that recognizes and binds to the antigen, e.g., a dysferlin polypeptide, but which does not substantially recognize and bind to other molecules in a sample (e.g., a biological sample) which naturally includes the antigen, e.g., a dysferlin polypeptide. An antibody that xe2x80x9cspecifically bindsxe2x80x9d to dysferlin is sufficient to detect a dysferlin polypeptide in a biological sample using one or more standard immunological techniques (for example, Western blotting or immunoprecipitation).
A xe2x80x9ctransgenexe2x80x9d is any piece of DNA, other than an intact chromosome, which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the host organism, or may represent a gene homologous to an endogenous gene of the organism.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The present materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. All the sequences disclosed in the sequence listing are meant to be double-stranded except the sequences of oligonucleotides.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.